The present invention relates to a method for determination of an analyte in a sample by using enzymatic Redox reaction, in which prior to the enzymatic Redox reaction, reducing substances in the sample are oxidized with hydrogen peroxide formed by another enzymatic Redox reaction in the presence of peroxidase.
It is known that a component in a biological sample can be determined by oxidizing the component with oxidase specific for the component in the presence of oxygen to form hydrogen peroxide and determining the formed hydrogen peroxide by a method known per se.
It is also known that when the component cannot be directly oxidized, the component is converted to a compound which can be directly oxidized with oxidase and then the above method is applied to the compound.
The determination of hydrogen peroxide is usually carried out by allowing the hydrogen peroxide to react with a chromogen in the presence of peroxidase to form a pigment and measuring the absorbance of the reaction mixture colored by the formation of the pigment.
The method described above is susceptible to interference from various substances present in the biological sample, for example, reducing substances such as bilirubin, cysteine, glutathione and medicines administered.
Especially, interference from reducing substances such as bilirubin (both conjugated and free types) is serious, which makes it difficult to accurately determine the component.
In order to reduce the influence of such reducing substances, studies have been made on methods in which an enzyme capable of decomposing reducing substances, e.g., bilirubin oxidase, is utilized. However, such methods have the problem that a long period of time is required for the decomposition of the reducing substances.
It is also known that hydrogen peroxide is effective for the decomposition of reducing substances [Bulletin of the Society of Clinical and Hygienic Assayers in Kyoto Prefecture, Japan, Vol. 10, No. 2, p. 31-36 (1983)]. However, when hydrogen peroxide is added directly to a sample, there occurs undesirable decomposition of the component to be determined due to the excessively strong oxidizing power of hydrogen peroxide. In addition to this, the use of hydrogen peroxide is not suitable for the production of an assay composition in the form of a marketable kit.
No method has thus far been found which enables avoidance of the undesirable interference from reducing substances contained in a sample and which is suitable for the production of a marketable assay kit.
As a result of studies on the decomposition of reducing substances in a biological sample, it has been found that the utilization of hydrogen peroxide formed by enzymatic Redox reaction using a component in the sample other than the component to be determined is very effective and does not involve the problems described above.
In this connection, U.S. Pat. No. 4,416,982 discloses a method for determination of an analyte which can be converted by the action of an enzyme (B) to the compound (A) which can be directly oxidized by the action of an oxidase capable of oxidizing the compound (A), which comprises the following steps:
(1) compound (A) in the original sample is oxidized by the action of the oxidase to form hydrogen peroxide;
(2) the resultant hydrogen peroxide is decomposed by adding peroxidase and phenol, aniline or derivatives thereof;
(3) the analyte is converted to compound (A) by the action of enzyme (B);
(4) the resultant compound (A) is oxidized by the action of the oxidase to form hydrogen peroxide; and
(5) the resultant hydrogen peroxide is determined by a known method.
The U.S. patent is silent about the decomposition of reducing substances. On the other hand, in the present invention, a component which does not perticipate in enzymatic Redox reaction using the analyte is used for the decomposition of reducing substances as described below.